Specific inhibition of the E.coli RecBCD enzyme by Chi sequences in single-stranded oligodeoxyribonucleotides

RecBCD is an ATP-dependent helicase and exonuclease which generates 3′ single-stranded DNA (ssDNA) ends used by RecA for homologous recombination. The exonuclease activity is altered when RecBCD encounters a Chi sequence (5′-GCTGGTGG-3′) in double-stranded DNA (ds DNA), an event critical to the generation of the 3′-ssDNA. This study tests the effect of ssDNA oligonucleotides having a Chi sequence (Chi⁺) or a single base change that abolishes the Chi sequence (Chi^o), on the enzymatic activities of RecBCD. Our results show that a 14 and a 20mer with Chi⁺ in the center of the molecule inhibit the exonuclease and helicase activities of RecBCD to a greater extent than the corresponding Chi^o oligonucleotides. Oligonucleotides with the Chi sequence at one end, or the Chi sequence alone in an 8mer, failed to show Chi-specific inhibition of RecBCD. Thus, Chi recognition requires that Chi be flanked by DNA at either end. Further experiments indicated that the oligonucleotides inhibit RecBCD from binding to its dsDNA substrate. These results suggest that a specific site for Chi recognition exists on RecBCD, which binds Chi with greater affinity than a non-Chi sequence and is probably adjacent to non-specific DNA binding sites.     

Stability of Lysozyme in Aqueous Extremolyte Solutions during Heat Shock and Accelerated Thermal Conditions

The purpose of this study was to investigate the stability of lysozyme in aqueous solutions in the presence of various extremolytes (betaine, hydroxyectoine, trehalose, ectoine, and firoin) under different stress conditions. The stability of lysozyme was determined by Nile red Fluorescence Spectroscopy and a bioactivity assay. During heat shock (10 min at 70°C), betaine, trehalose, ectoin and firoin protected lysozyme against inactivation while hydroxyectoine, did not have a significant effect. During accelerated thermal conditions (4 weeks at 55°C), firoin also acted as a stabilizer. In contrast, betaine, hydroxyectoine, trehalose and ectoine destabilized lysozyme under this condition. These findings surprisingly indicate that some extremolytes can stabilize a protein under certain stress conditions but destabilize the same protein under other stress conditions. Therefore it is suggested that for the screening extremolytes to be used for protein stabilization, an appropriate storage conditions should also be taken into account.     

Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.     

Liver X receptor preferentially activates de novo lipogenesis in human preadipocytes

The liver X receptor (LXR) was demonstrated to play a key role in cholesterol metabolism in liver, intestine and macrophage. However, its function on the regulation of preadipocyte differentiation remains unclear since contradictory results were reported. The objective of the present study was to unravel the functionality of LXR in human preadipocytes. We show that the LXR agonist T0901317 strongly stimulated the expression of SREBP-1c and the lipogenic enzymes ACC-1, FAS and SCD-1 in both the human preadipose cell line Chub-S7 as well as human primary stromal vascular fraction (SVF) cells. The effects on gene expression were associated with the stimulation of de novo lipogenesis in both cell models, resulting in the induction of lipid accumulation. In contrast with a PPARgamma agonist (BRL49653), T0901317 enhanced only slightly the expression of PPARgamma dependent genes (PPARgamma, aP2 and adiponectin) in Chub-S7 cells and failed to change their expression in human SVF cells. These results show that LXR stimulated preferentially triglyceride accumulation in human preadipocytes via the induction of de novo lipogenesis, rather than activating the differentiation process through PPARgamma activation.     

Functional capacity of XRCC1 protein variants identified in DNA repair-deficient Chinese hamster ovary cell lines and the human population

XRCC1 operates as a scaffold protein in base excision repair, a pathway that copes with base and sugar damage in DNA. Studies using recombinant XRCC1 proteins revealed that: a C389Y substitution, responsible for the repair defects of the EM-C11 CHO cell line, caused protein instability; a V86R mutation abolished the interaction with POLβ, but did not disrupt the interactions with PARP-1, LIG3α and PCNA; and an E98K substitution, identified in EM-C12, reduced protein integrity, marginally destabilized the POLβ interaction, and slightly enhanced DNA binding. Two rare (P161L and Y576S) and two frequent (R194W and R399Q) amino acid population variants had little or no effect on XRCC1 protein stability or the interactions with POLβ, PARP-1, LIG3α, PCNA or DNA. One common population variant (R280H) had no pronounced effect on the interactions with POLβ, PARP-1, LIG3α and PCNA, but did reduce DNA-binding ability. When expressed in HeLa cells, the XRCC1 variants—excluding E98K, which was largely nucleolar, and C389Y, which exhibited reduced expression—exhibited normal nuclear distribution. Most of the protein variants, including the V86R POLβ-interaction mutant, displayed normal relocalization kinetics to/from sites of laser-induced DNA damage: except for E98K and C389Y, and the polymorphic variant R280H, which exhibited a slightly shorter retention time at DNA breaks.     

Behavioral abnormalities in captive nonhuman primates

In this study, we dealt with 11 species of nonhuman primates across 10 zoos in India. We recorded behavior as instantaneous scans between 9 a.m. and 5 p.m. In the study, we segregated behaviors for analyses into abnormal, undesirable, active, and resting. The 4 types of abnormal behavior exhibited included floating limb, self-biting, self-clasping, and stereotypic pacing. In the study, we recorded 2 types of undesirable behavior: autoerotic stimulation and begging. Langurs and group-housed macaques did not exhibit undesirable behaviors. A male lion-tailed macaque and a male gibbon exhibited begging behavior. autoerotic stimulation and self-biting occurred rarely. Males exhibited higher levels of undesirable behavior than did females. Animals confiscated from touring zoos, circuses, and animal traders exhibited higher levels of abnormal behaviors than did animals reared in larger, recognized zoos. The stump-tailed macaque was the only species to exhibit floating limb, autoerotic stimulation, self-biting, and self-clasping. Our results show that rearing experience and group composition influence the proportions of abnormal behavior exhibited by nonhuman primates in captivity. The history of early social and environmental deprivation in these species of captive nonhuman primates probably is critical in the development of behavioral pathologies. Establishing this will require further research.     

Enclosure design and space utilization by Indian leopards (Panthera pardus) in four zoos in Southern India

Enclosure design and the use of enclosure space influence the activity budget of captive leopards. The study laid out in grids all enclosures on the base map and segregated these grids into 4 zones. Every 5 min, the study recorded the proportion of time spent in these zones with the leopards' behavior. Captive leopards most frequently used the “edge ”zone. Almost all leopards used the edge zone for stereotypic pacing, the “back ”zone for resting, and the “other ”zone for activity. The study positively correlated the proportion of time spent in the “enriched ”zone with activity levels exhibited by leopards housed in some enclosures and with resting in others. Thus, the study segregated structural objects in the enriched zone into activity-related features (e.g., logs) and rest-related features (e.g., trees and sleeping platforms). Compared with individuals housed in barren enclosures, leopards housed in structurally enriched on-exhibit enclosures exhibited higher levels of activity. Enclosure design was found to be an important factor influencing the welfare of leopards in captivity.